Letter reSeArCH
Extended Data Fig. 6 | Interactions between CaM and Ca^2 + from the
crystal structures and the role of Ca^2 + in the activation of SidJ by CaM.
a, Key residues of CaM involved in the interaction with Ca^2 +. Ca^2 + is
coordinated by D21, D23, D25 and T27 of CaM, which are shown as red
sticks. Ca^2 + is shown as a pink sphere. Electron density of a simulated
annealing Fo − Fc omit map for Ca^2 + contoured at 3.0σ. b, Dialysis against
20 mM EGTA does not abolish the activity of SidJ. All proteins used in
the reactions were dialysed against a buffer containing 20 mM EGTA
for 14 h. SdeA was incubated with SidJ in reactions containing ATP
and EGTA-dialysed CaM for 2 h at 37 °C. Reactions without SidJ were
established as a control. A cocktail containing 4×Flag–Rab33b, NAD+ and
ubiquitin was added to each reaction. After further incubation for 2 h at
37 °C, proteins resolved by SDS–PAGE were analysed with the indicated
antibodies. SdeA activity was measured by the production of ubiquitinated
Rab33b as indicated by a shift in molecular mass. c, The activity of SidJ
is not sensitive to 10 mM EGTA. SdeA was first incubated with SidJ for
glutamylation with the indicated amounts of EGTA for 2 h at 37 °C. NAD+,
4 ×Flag–Rab33b and ubiquitin were then supplemented to the reactions,
which were allowed to proceed for 2 h at 37 °C before resolution by SDS–
PAGE. Rab33b modification was detected as described in b. Proteins in the
reactions were detected by immunoblotting with specific antibodies. In
b, c, similar results were obtained in at least three independent experiments.